Natural and engineered neutralizing antibodies for universal protection against ebolaviruses

Abstract

Ebola virus (EBOV) and related filoviruses are the causative agents of sporadic outbreaks of highly lethal hemorrhagic fever in Middle and West Africa. To date, no Food & Drug Administration (FDA) approved therapeutics or vaccines are available for treatment of filovirus infections. The vast 2013-2016 EBOV outbreak in West Africa underscored the urgent need for antiviral treatments and demonstrated the potential of passive immunotherapy to reverse advanced Ebola virus disease (EVD). However, existing monoclonal antibody (mAb) cocktails such as ZMapp(TM) are limited by a narrow spectrum of antiviral activity, which stems from viral strain-specific neutralization of the highly variable entry glycoprotein (GP) by most mAbs. This thesis work comprises two projects aimed at discovery and development of broadly neutralizing antibodies as therapeutic candidates for pan-ebolavirus protection.;GP, the sole viral protein displayed on the surface of filovirus particles, is necessary and sufficient for entry. Following attachment and uptake into the cell, filovirus particles are trafficked though the endocytic compartments to late endosomes and lysosomes. Endosomal acid-dependent cysteine proteases prime the viral GP into a fusion-competent state by removing a large portion of the protein, thus exposing the recessed receptor binding site (RBS). Productive infection by all filoviruses, including EBOV, requires the subsequent interaction with their conserved entry receptor, the endo/lysosomal membrane protein Niemann-Pick Cl (NPC1). We reasoned that targeting this obligate interaction with antibodies could confer broad and potent antiviral activity. Unfortunately, conventional Abs cannot target the intracellular sites of GP-NPC1 engagement because they are excluded from endosomes.;The first part of this dissertation describes our work on engineered bispecific antibody (bsAb) molecules targeting either NPC1, or the cryptic but conserved receptor binding site in ebolavirus glycoproteins, both present only inside cellular endosomes. These "Trojan horse" bispecific antibodies exploit virus particles themselves for delivery to the endosomal sites of virus-receptor interaction. The bsAbs broadly and potently neutralized infection of multiple ebolaviruses in vitro in a manner that required both their engagement of GP in extracellular virions and intracellular blockade of the GP-NPC1 interaction. In vivo studies in mouse models of EBOV and Sudan virus (SUDV) infection demonstrated that a single bsAb targeting the intracellular virus-receptor interface can afford broad protection against divergent ebolaviruses.;In a complementary body of work, the focus of the second part of this dissertation, we mined a monoclonal antibody library from a human survivor of Ebola virus infection to identify naturally occurring broadly neutralizing antibodies. Our work revealed that antibodies with pan-ebolavirus neutralizing activity arose in this individual with low frequency and were distinguished by exceptional neutralization potency, despite having undergone minimal levels of somatic hypermutation. We found that these mAbs recognize an inter-protomer epitope within the fusion loop, an essential and conserved component of the viral membrane fusion machinery. We also demonstrated that these mAbs act though a novel mechanism that involves targeting and neutralization of an endosomal, fusion-competent viral intermediate during entry. In animal challenge studies, we observed strong protection in mouse and ferret models of infection with three divergent ebolaviruses mediated by these pan-ebolavirus neutralizers. Removal of a conserved, fusion loop proximal N-linked glycan from EBOV GP further potentiated the neutralizing activity of our pan-ebolavirus mAbs. This in turn suggests that suitably engineered immunogens could help elicit potent and broad neutralizers of this kind with higher frequency and points the way for improved vaccine development.